There are over 240 Type II restriction endonucleases (REases) with unique specificities discovered so far from bacterial and viral sources. In contrast, only eight site-specific nicking enzymes are commercially available. Nonetheless, nicking endonucleases are believed to be useful in a variety of contexts including strand displacement amplification and DNA cloning (U.S. Pat. No. 6,660,475; EP1176204A1; WO 03/087301).
Certain sequence-specific DNA nicking enzymes have been found to occur naturally. Nt.CviQXI (CviNY2A, R^AG) and Nt.CviPII (CviNYSI, ^CC(A/G/T)) were originally found in the lysates of Chlorella viruses (Xia, Y. et al. Nucl Acids Res. 16:9477-9487, (1988); Zhang Y. N. M. et al. Virology 240:366-375 (1998). The nicking enzymes N.BstSEI and N.BstNBI were identified in bacterial sources (Abdurashitov M. A. et al. Mol Biol. (Mosk) 30:1261-1267, (1996); Morgan R. D. et al. Biol. Chem. 381:1123-1125 (2000)). Bacteriophages also encode nicking enzymes such as the gene II protein of bacteriophage f1 that is essential for viral DNA replication (Geider K. et al. Advan. Expt. Med. Biol. 179:45-54 (1984)).
Sequence-specific DNA nicking enzymes have also been created by mutating naturally occurring dimeric Type IIA, Type IIS (Xu Y. et al. Proc. Natl. Acad. Sci. USA 98:12990-12995 (2001); Besnier C. E. et al. EMBL Rep. 2:782-786 (2001); Zhu Z. et al. J. Mol. Biol. 337:573-583 (2004)) or Type IIT restriction endonucleases using a variety of approaches.
Type II restriction endonucleases (REase) generally have two subunits forming either a homodimer or a heterodimer.
For homodimeric EcoRV, Stahl et al. (Proc Natl Acad Sci USA 93(12):6175-80 (1996)) described combining a subunit with an inactive catalytic activity with a second subunit with a deficiency in DNA binding to produce a nicking endonuclease that is non-specific with respect to which strand is nicked.
Heitman (Mol. Microbiol 33:1141-1151 (1999)) created EcoRI nicking endonucleases with R200C, R200K, or E144C mutations.
Xu (Proc. Natl. Acad. Sci. USA 98:12990-12995 (2001)) reported the creation of N.AlwI by domain exchange between the Type IIS REase AlwI and a homologous, naturally occurring nicking enzyme, N.BstNBI. This nicking endonuclease predominantly nicks the top DNA strand of a DNA duplex as a monomer. This domain exchange method requires prior knowledge of the dimerization domain and a relatively high amino acid sequence similarity with a naturally existing nicking enzyme.
Site-directed mutagenesis of MlyI REase resulted in variants in which the dimerization function was disrupted. The resulting nicking enzyme is strand-specific, cleaving the top strand of the wild type recognition sequence. However, no bottom strand nicking enzyme was ever isolated from MlyI (Besnier C. E. et al. EMBL Rep. 2:782-786 (2001).
The DNA nicking activity of BfiI can be enhanced by alteration of reaction conditions. By lowering the pH value in the cleavage reaction, the BfiI REase can be converted to a bottom-strand specific nicking enzyme (Sasnauskas G. et al. Proc. Natl. Acad. Sci. USA 100:6410-6415 (2003)).
Zhu (J. Mol. Biol. 337:573-583 (2004)) used random mutagenesis and back-crosses with the Type IIS restriction endonucleases to generate BsaI, BsmAI and BsmBI nicking variants. There was no selectivity in nicking strand specificity. The random mutagenesis method required screening a large number of mutants.
Samuelson (Nucl. Acids Res. 32:3661-3671 (2004)) designed a SapI substrate site into the expression plasmid to allow for in vitro selection of plasmid clones from a randomly mutated SapI expression library possessing a site-specific and strand-specific nick. Bottom-strand nicking enzymes yielded Nb.SapI-1 containing a critical R420I substitution near the end of the protein while a separate top-strand selection procedure yielded several SapI variants with a distinct preference for top-strand cleavage.
Nicking endonucleases have been created from heterodimeric Type IIT including Bpu10I (Stankevicius K. et al. Nucl. Acids Res. 26:1084-1091 (1998), EP 1176204 A1, July 2000, BbvCI (US patent application 2003/0100094 and BslI (Hsieh et al. J. Bacteriol. 182:949-955 (2000)) These nicking endonucleases were formed by inactivation of the catalytic activity of one subunit in the heterodimer.
Nicking BsmAI and BsmB1 have been made by error prone PCR and site directed mutagenesis (U.S. application Ser. No. 11/013,235).